Mesenchymal stem cells (MSCs) are an exciting cell-based therapeutic candidate for cardiovascular disease with demonstrated efficacy in regenerating damaged heart tissue through secretion of trophic factors that promote angiogenesis and cardiomyogenesis. Significant strides have been made in deciphering the molecules in the MSC secretome involved in repair; however, the cues in the MSC microenvironment that direct secretion are not understood. In the proposed work we will investigate how the properties of the extracellular matrix direct pro-angiogenic signaling using MSCs and human microvascular endothelial cells (HMVECs) as a model heterotypic system. This work is important because the physical and chemical properties of the matrix have been demonstrated to influence MSC secretion of pro-angiogenic factors. Although we now understand many of the mechanisms responsible for MSC therapeutic efficacy, current methods used in clinical trials do not provide control over these pathways. In the first aim we will use our self- assembled monolayer chemistry to micropattern MSCs in geometries that have been demonstrated to promote paracrine signaling. By controlling the cytoskeletal architecture in single cells we will explore how mechanotransduction influences signaling pathways that regulate secretion of trophic factors. In the second aim, we will explore the influence of matrix mechanics and composition by immobilizing fibronectin, vitronectin, laminin and collagen to polyacrylamide gels across a range of physiologically relevant mechanical properties. We will combine geometry with matrix compliance and composition to study potential synergies between these cues in guiding pro-angiogenic signaling. Conditioned media from these experiments will be added to matrigel cultures of HMVECs to quantitate the degree of tubulogenesis. We will use blocking antibodies, siRNA and pharmacological inhibition strategies to modulate mechanotransduction pathways in MSCs cultured under these conditions to evaluate the link between extracellular signaling and paracrine secretion. Antibody arrays will be used to profile the pro-angiogenic secretome stimulated by mechanochemical signals in the microenvironment. In the final aim, we will fabricate a heterotypic co-culture system that will enable the translation of these physical and biochemical cues to a 3D model system that is more amenable to the development of clinically viable biomaterials. This work will lead to an improved understanding of the mechanical and biochemical components of the extracellular environment that promotes pro-angiogenic signaling from MSCs. Understanding the cues in the microenvironment that direct MSC secretion-and implementing these criteria into the design of biomaterials-will be critical for the development of efficacious cell-based therapeutic strategies.